Proper functioning of the cell cycle and its regulation in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Major control over cell cycle progression is exerted by ubiquitin- mediated proteolysis. For instance, the initiation of chromosome separation (anaphase) and completion of the cell cycle require the degradation of a number of cell cycle regulatory proteins such as cyclins, securin (which regulates the protein holding chromosomes together), and microtubule motor proteins. The ubiquitin ligase (or E3) for this proteolysis is termed the Anaphase Promoting Complex (APC). Proteins are targeted to the APC by two substrate recognition proteins, Cdc20 and Cdh1, active during late mitosis and in G1, respectively. An emerging theme in the regulation of the APC involves the actions of pseudosubstrates, proteins that are not themselves APC substrates but that compete with substrates for binding to Cdc20 and Cdh1 via the Destruction Box and KEN Box sequences found in APC substrates. Acm1 is a recently- identified pseudosubstrate inhibitor of APCCdh1 in budding yeast. In addition to a Destruction Box and a KEN box, Acm1 contains a novel motif that facilitates its interaction with Cdh1. Acm1 is also subject to ubiquitin-mediated proteolysis, both by APCCdc20 during mitosis and by an unidentified mechanism throughout the cell cycle, and is protected from both pathways via phosphorylation. To further our understanding of APC regulation via pseudosubstrate inhibition, we propose the following Specific Aims: 1) To determine the Acm1 motifs and cellular pathways responsible for Acm1 degradation and the role(s) of Acm1 phosphorylation in protecting it from degradation. 2) To use genetic screens to identify novel human and yeast pseudosubstrate APC inhibitors. We will also further explore the mechanism by which the vertebrate Emi1 protein acts as a pseudosubstrate inhibitor.